Min Qi, Yang Lu, Wang Yu, Liu Yili, Jiang Mingfeng
Institute of Qinghai-Tibetan Plateau, Southwest Minzu University, Chengdu 610041, China.
College of Animal and Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China.
Animals (Basel). 2023 Mar 3;13(5):925. doi: 10.3390/ani13050925.
Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative C method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that , and are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining , and as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle.
牦牛胃中的高效营养吸收和能量代谢有助于它们适应恶劣环境。准确的基因表达谱分析将有助于进一步揭示牦牛胃中营养和能量代谢的分子机制。逆转录定量聚合酶链反应（RT-qPCR）被认为是分析基因表达的一种准确可靠的方法。参考基因的选择对于获得有意义的RT-qPCR结果至关重要，尤其是在组织和器官的纵向基因表达研究中。我们的目标是从转录组中选择并验证最佳参考基因，作为牦牛胃纵向基因表达研究的内参。在本研究中，根据转录组测序（RNA-seq）结果和先前的文献确定了15个候选参考基因（CRGs）。使用RT-qPCR对这15个CRGs在牦牛胃中的表达水平进行定量，包括瘤胃、网胃、瓣胃和皱胃在五个阶段的表达：0天、20天、60天、15个月和3岁（成年）。随后，通过四种算法评估这15个CRGs的表达稳定性：geNorm、NormFinder、BestKeeper和比较Cq法。此外，使用RefFinder获得CRGs稳定性的综合排名。分析结果表明，[具体基因1]、[具体基因2]和[具体基因3]是牦牛胃在整个生长周期中最稳定的基因。此外，为了验证所选CRGs的可靠性，使用三个最稳定或三个最不稳定的CRGs通过RT-qPCR对[目标基因]的相对表达水平进行定量。总体而言，我们建议将[具体基因1]、[具体基因2]和[具体基因3]组合作为参考基因，用于牦牛胃在整个生长周期中RT-qPCR数据的标准化。
